WO2010053316A2 - New use of sixth immunoglobulin-like domain of vcam-1 - Google Patents

Abstract

The present invention relates to a use of the sixth immunoglobulin-like domain of vascular cell adhesion molecule-1 (VCAM-1), and more particularly, relates to a method of inhibiting activity of the sixth immunoglobulin-like domain of VCAM-1 to inhibit the transmigration of leukocyte across endothelial cell, and thereby preventing or treating VCAM-1 mediated-diseases using the same. According to the present invention, it is expected that it will be possible not only to screen materials that can inhibit the transmigration of leukocyte across endothelial cell without affecting the binding of leukocyte to endothelial cell, but also to effectively prevent or treat VCAM-1 mediated-diseases.

Description

NEW USE OF SIXTH IMMUNOGLOBULIN-LIKE DOMAIN OF VCAM-1

The present invention relates to a use of the sixth immunoglobulin-like domain of vascular cell adhesion molecule-1 (VCAM-1), and more particularly, relates to a method of inhibiting activity of the sixth immunoglobulin-like domain of VCAM-1 to inhibit the transmigration of leukocyte across endothelial cell, and thereby preventing or treating VCAM-1 mediated-diseases using the same.

In inflammatory responses, cell adhesion molecules play a key role in promoting the binding of leukocytes or lymphocytes to activated endothelium, allowing transmigration and then, ultimately, inducing severe damage to cells or tissues. In response to inflammatory cytokines, endothelial cells up-regulate the expression of various adhesion molecules, such as E- and P-selectins and members of the immunoglobulin superfamily including intercellular cell adhesion molecule (ICAM)-1, -2, and -3, and vascular cell adhesion molecule (VCAM-1).

Among these, VCAM-1 (CD106) is dominantly and inducibly expressed on endothelial cells upon activation by lipopolysaccharide (LPS), interleukin-1 (IL-1), interferon-γ (IFNγ), hydrogen peroxide (H₂O₂) or tumor necrosis factor alpha (TNFα).

Many increasing reports have been suggested the potential role of VCAM-1 to be closely involved in the progression of various inflammatory disorders including atherosclerosis and rheumatoid arthritis. Furthermore, by us and other colleagues, the possible relevance of VCAM-1 on inflammatory response in organ- or islet transplantation has been also suggested.

According to many increasing reports, VCAM-1 and α4β1 integrin play a key role in leukocyte trafficking of many inflammatory responses. Although so far much attention has been paid to α4β1 integrin as a therapeutic intervention for inflammatory disorders, however, currently, VCAM-1 may be a better molecular target for generating therapeutic antibody for the following reasons: First, Natalizumab, an antibody to α4β1 integrin, was initially reported to be effective in the prevention of experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis, but has proven to have the risk of developing proessive multifocal leukoencephalophathy. Second, α4β1 integrin has many binding partners other than VCAM-1, like paxillin, osteopontin, thrombospondin-1, fibronectin, and junctional adhesion molecule-2 (JAM-2). Due to this, application of anti-α4β1 integrin antibody may be relatively incomplete in suppressing inflammatory responses or cause certain side effects. Third, VCAM-1 is exclusively expressed on activated endothelial cells whereas leukocytes/lymphocytes expressing α4β1 integrin exist in whole human body through blood stream. Thus, it is expected that anti-VCAM-1 antibody can be locally effective to inflamed endothelial cells with inflammation among blood vessels. Therefore, developing antibody specific to VCAM-1 may be critical for specifically alleviating VCAM-1-mediated inflammatory responses.

VCAM-1 is critical for the progression of many inflammatory disorders including atherosclerosis, rheumatoid arthritis, and transplantation rejection. Despite of much progress about the functional role of VCAM-1, so far, most antibodies specific to VCAM-1 have a limit to lack broad cross-species reactivity. For example, although rat anti-mVCAM-1 specific mAb (clone MK2.7) has been demonstrated to reduce collagen-induced arthritis and islet allograft rejection, it is only specific to mouse VCAM-1. Other mouse anti-hVCAM-1 specific mAb (clone 1.4C3) was also reported. To this end, the development of a VCAM-1 specific mAb having broad cross-species reactivity is required to simultaneously crystallize information from various in vitro and animal studies.

VCAM-1 consists of extracellular domain containing seven immunoglobulin (IgG)-like domains, transmembrane domain, and cytosolic domain. Especially, among seven IgG-like domains of VCAM-1, the first and fourth IgG-like domains in the extracellular domain are important for binding to very late antigen-4 (VLA-4), α4β1 integrin, expressed on monocytes/macrophages or T lymphocytes in various inflammatory responses. However, so far, it has not clearly identified about the functional role of the other IgG-like domains of VCAM-1 domain yet. So far, many studies have focused on the functional significance of the interaction between the first and fourth homologous domains of VCAM-1 and α4β1 integrin in inflammatory responses. However, this interaction has been also known to be closely involved in normal physiology including B cell development, tissue regeneration by stem cells, and muscle development. In this regards, the identification of the other domain of VCAM-1 not inhibiting both interactions but leukocytes/lymphocytes trafficking may be more essential for therapeutic intervention in inflammatory responses.

In the present study, we for the first time developed anti-VCAM-1 mAb specific to the sixth IgG-like domain of VCAM-1 using phage display technology. With this mAb, we found that the sixth IgG-like domain is important for the transmigration of U937 human monocytic cell lines across activated HUVECs, regardless of the interaction between α4β1 integrin and VCAM-1. Finally, we also demonstrated that the with mouse islet allograft model, the sixth IgG-like domain could be a new molecular target for alleviating inflammatory responses in vivo. In summary, this study suggests that the sixth IgG-like domain of VCAM-1 may be a new therapeutic target for alleviating VCAM-1-mediated inflammatory responses.

A purpose of the present invention is to provide a method for inhibiting the transmigration of leukocyte across endothelial cell by inhibiting activity of the sixth immunoglobulin-like domain of vascular cell adhesion molecule-1 (VCAM-1).

Another purpose of the present invention is to provide a method of preventing or treating VCAM-1 mediated-diseases by inhibiting activity of the sixth immunoglobulin-like domain of vascular cell adhesion molecule-1 (VCAM-1).

Another purpose of the present invention is to provide a kit for screening materials treating or preventing VCAM-1 mediated-diseases, comprising the sixth immunoglobulin-like domain of vascular cell adhesion molecule-1 (VCAM-1) and a kit for the same.

Another purpose of the present invention is to provide a method of screening VCAM-1 mediated-diseases treatment or prevention materials, comprising the steps of: (1) contacting a patient of VCAM-1 mediated-diseases patient with a sample; (2) detecting increase or decrease of activity of the sixth immunoglobulin-like domain of vascular cell adhesion molecule-1 (VCAM-1) expressed on activated endothelial cell; and (3) determining the sample that inhibits activity as a VCAM-1 mediated-diseases treatment or prevention materials.

Hereinafter, the present invention is explained in more detail.

As an aspect for achieving the above purposes, the present invention provides A method for inhibiting the transmigration of leukocyte across endothelial cell by inhibiting activity of the sixth immunoglobulin-like domain of vascular cell adhesion molecule-1 (VCAM-1).

It should be understood that the definitions of terms used hereunder are provided to more clearly explain the aspects of the present invention, and to depict embodiments of specific aspects, and thus do not regarded as being used for limiting the present invention.

Various definitions are used throughout this document. Most words have the meaning that would be attributed to those words by one skilled in the art. Words specifically defined either below or elsewhere in this document have the meaning provided in the context of the present invention as a whole and as are typically understood by those skilled in the art.

The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, molecular biology, immunology and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.); and Handbook of Experimental Immunology, VoIs. I-IV (D.M. Weir and CC. Blackwell, eds., 1986, Blackwell Scientific Publications); and Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989).

As used herein, the singular forms "a," "an" and "the" include plural references unless the content clearly dictates otherwise. Thus, for example, reference to "an antibody" includes a mixture of two or more such antibodies.

As used herein, the term "about" refers to +/- 20%, +/- 10%, or +/- 5% of a value.

The term "domain of VCAM-1" as used herein refers to a structural part of a biomolecule that contributes to a function of the VCAM-1. Domains may be coextensive with regions or portions thereof and may also incorporate a portion of a biomolecule that is distinct from a particular region, in addition to all or part of that region.

The term "region" refers to. a physically contiguous portion of the primary structure of a biomolecule. In the case of proteins, a region is defined by a contiguous portion of the amino acid sequence of that protein, hi some embodiments a "region" is associated with a function of the biomolecule.

The term “the sixth immunoglobulin-like domain of vascular cell adhesion molecule-1” as used herein refers to comprising a domain located at the sixth counted from N-terminal among seven domains identified in VCAM-1, 0-127 proteins of N-terminal around that, and 0-117 proteins of C-terminal around that.

The amino acid sequence of domain located at the sixth counted from N-terminal among seven domains identified in human VCAM-1 sets forth in SEQ NO. 5. And, the nucleic acid of domain located at the sixth counted from N-terminal among seven domains identified in human VCAM-1 sets forth in SEQ NO. 6.

The amino acid sequence of domain located at the sixth counted from N-terminal among seven domains identified in mouse VCAM-1 sets forth in SEQ NO. 7. And, the nucleic acid of domain located at the sixth counted from N-terminal among seven domains identified in mouse VCAM-1 sets forth in SEQ NO. 8.

Preferably, the sixth immunoglobulin-like domain of VCAM-1 may be derived from human and comprise amino acid sequence of SEQ NO. 1. More preferably, sixth immunoglobulin-like domain of VCAM-1 may be described as amino acid sequence of SEQ NO. 1. Most preferably, the sixth immunoglobulin-like domain of VCAM-1 may be encoded by nucleotide of SEQ NO. 2.

Preferably, the sixth immunoglobulin-like domain of VCAM-1 may be derived from mouse and comprise amino acid sequence of SEQ NO. 3. More preferably, sixth immunoglobulin-like domain of VCAM-1 may be described as amino acid sequence of SEQ NO. 3. Most preferably, the sixth immunoglobulin-like domain of VCAM-1 may be encoded by nucleotide of SEQ NO. 4.

The term "fragment" as used herein refers to a physically contiguous portion of the primary structure of a biomolecule. In the case of proteins, a portion is defined by a contiguous portion of the amino acid sequence of that protein and refers to at least 3-5 amino acids, at least 8-10 amino acids, at least 11-15 amino acids, at least 17-24 amino acids, at least 25-30 amino acids, and at least 30-45 amino acids. In the case of oligonucleotides, a portion is defined by a contiguous portion of the nucleic acid sequence of that oligonucleotide and refers to at least 9-15 nucleotides, at least 18-30 nucleotides,; at least 33-45 nucleotides, at least 48-72 nucleotides, at least 75-90 nucleotides, and at least 90-130 nucleotides.

"Inhibiting activity of the sixth immunoglobulin-like domain of VCAM-1", as used herein, refers to an decrease in activity of the sixth immunoglobulin-like domain of VCAM-1 that can be a result of, for example, interaction of an agent with a polynucleotide or polypeptide of the sixth immunoglobulin-like domain of VCAM-1, inhibition of transcription and/or translation of the sixth immunoglobulin-like domain of VCAM-1 (e.g., through antisense or siRNA interaction with the gene of the sixth immunoglobulin-like domain of VCAM-1 or transcript of the sixth immunoglobulin-like domain of VCAM-1, through modulation of transcription factors that inhibit expression of the sixth immunoglobulin-like domain of VCAM-1), and the like. For example, inhibition of a biological activity refers to a decrease in a biological activity.

As used herein, the term "inhibit" refers to a reduction, decrease, inactivation or down-regulation of an activity or quantity. For example, in the present invention, modulators of the sixth immunoglobulin-like domain of VCAM-1 can inhibit at least one of the transmigration of leukocyte across endothelial cell; VCAM-1 mediated signal after the transmigration of leukocyte across endothelial cell; generation, amplification, residue, transference or transplantation rejection of VCAM-1 mediated disease. Inhibition may be at least 25%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%, as compared to a control.

By inhibiting activity of the sixth immunoglobulin-like domain of VCAM-1, it enables to inhibit the transmigration of leukocyte across endothelial cell, however, preferably it does not affect the binding of leukocyte across endothelial cell.

The term “inhibiting activity of leukocyte across endothelial cell” refers to reduction or abolition of the transmigration of leukocyte across endothelial cell under the presence of activity inhibitor of the sixth immunoglobulin-like domain of VCAM-1 by at least 25%, 50%, 75%, 85%, 90%, 95%, or 100%. Inhibition of the transmigration of leukocyte across endothelial cell or the extent of the inhibition can be measured by assays containing transendothelial cell migration assay, hematoxylin and eosin staining and immunohistochemistry, but it will not be limited thereto and can be measured using various assays known in this art. By inhibiting the transmigration of leukocyte across endothelial cell, the signal progress after transmigration is inhibited, and one of the resulted effects include prevention and treatment of VCAM-1 mediated diseases.

In the above description, the phrase “does not affect the binding of leukocyte across endothelial cell” means that no change can be found in the binding and the extent thereof, when compared to a case where the activity of the sixth immunoglobulin-like domain of VCAM-1 is inhibited. It has the same meaning as the phrase “do not inhibit the binding of leukocyte to endothelial cell” below described. The binding can be measured using various methods known in this art, for example, by labeling leukocyte or labeling expression of integrin in leukocyte, and measuring the labeled extent on the endothelial cell using fluorescence plate reader or flow cytometry. In such a case, labeling can be performed using a fluorescent probe, such as, CFSE(carboxyfluorescein succinimidyl ester) or CFDA-SE(carboxyfluorescein diacetate succinimidyl ester), but it will not be limited thereto.

In an embodiment of the present invention,

To investigate the role of the sixth IgG-like domain of VCAM-1 on the transmigration of leukocyte, HUVECs(human umbilical vein endothelial cells) plated on the transwell were incubated in the absence or presence of hTNFα(human tumor necrosis factor alpha). Then, transendothelial cell migration assay was performed with U937 human monocytic cell lines in the absence or presence of anti-VCAM-1 Fab. As shown in Fig. 3, inventors found that anti-VCAM-1 mAb could significantly inhibit the transmigration of U937 cells across activated HUVECs.

To further verify the role of the sixth IgG-like domain on VCAM-1-α4β1 interaction, we first generated vector- or hVCAM-1-overexpressing HEK 293 cell lines (Fig. 4A). Then, inventors performed neutralizing assay with those HEK293 cell lines, HAEC(human aortic endotheluial cells), and HUVEC and CSFE-labeled U937 in the absence or presence of anti-VCAM-1 mAb. As shown in Fig. 4B-4C, inventors found that this mAb little affect the VCAM-1-mediated binding of leukocytes to activated endothelial cells.

Inhibition of activity of the sixth immunoglobulin-like domain of VCAM-1 can be accomplished by a modulator of the sixth immunoglobulin-like domain of VCAM-1.

In some embodiments, modulator of the sixth immunoglobulin-like domain of VCAM-1 comprises an oligonucleotide, a small molecule, a chemical compound, a decoy, or an antibody. In some embodiments, modulator of the sixth immunoglobulin-like domain of VCAM-1 inhibits a biological activity of the sixth immunoglobulin-like domain of VCAM-1 by 25%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, 97%, 98%, 99% or 100%, as compared to a control. In some embodiments, modulator of the sixth immunoglobulin-like domain of VCAM-1 inhibits expression of sixth immunoglobulin-like domain of VCAM-1 by at least 25%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, 97%, 98%, 99% or 100%, as compared to a control.

As used herein, the term "modulating" refers to a change in the quality or quantity of a gene, protein, or any molecule that is inside, outside, or on the surface of a cell. The change can be an increase or decrease in expression or level of the molecule. The, term "modulates" also includes changing the quality or quantity of a biological function/activity.

As used herein, the term "modulator" refers to a composition that modulates one or more physiological or biochemical events associated with VCAM-1 mediated-diseases. In some embodiments the modulator inhibits one or more biological activities associated with VCAM-1 mediated-diseases. In some embodiments the modulator is a small molecule, an antibody, a chemical compound, a decoy or an oligonucleotide. In some embodiments the modulator acts by blocking ligand binding or by competing for a ligand-binding site. In some embodiments the modulator acts independently of ligand binding. In some embodiments the moulators can block or compete VCAM-1 complex formation that can lead to the activation of VCAM-1 downstream signaling. The modulators inhibit activity of the sixth immunoglobulin-like domain of VCAM-1 and thereby inhibiting at least one of the transmigration of leukocyte across endothelial cell; VCAM-1 mediated signal after the transmigration of leukocyte across endothelial cell; generation, amplification, residue, transference or transplantation rejection of VCAM-1 mediated disease, or inhibiting expression of the sixth immunoglobulin-like domain of VCAM-1.

As used herein, the term "antibody" refers to monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies, bifunctional/bispecif[iota]c antibodies, humanized antibodies, human antibodies, and complementary determining region (CDR)- grafted antibodies, that are specific for the target protein or fragments thereof. The term "antibody" further includes in vivo therapeutic antibody gene transfer. Antibody fragments, including Fab, Fab ', F(ab ')2, . scFv,- and Fv are also provided by the invention.

As used herein, the term "epitope" refers to an antigenic determinant of a polypeptide. In some embodiments an epitope may comprise 3 or more amino acids in a spatial conformation which is unique to the epitope. In some embodiments epitopes are linear or conformational epitopes. Generally an epitope consists of at least 4, at least 6, at least 8, at least 10, and at least 12 such amino acids, and more usually, consists of at least 8-10 such amino acids. Methods of determining the spatial conformation of amino acids are known in the art, and include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance.

In some embodiments modulator of the sixth immunoglobulin-like domain of VCAM-1 is a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a human antibody, a humanized antibody, a single-chain antibody, or a Fab fragment. The antibody may be labeled with, for example, an enzyme, radioisotope, or fluorophore.

The invention also provides antibodies that competitively inhibit binding of an antibody to an epitope of the invention as determined by any method known in the art for determining competitive binding using, for example, immunoassays. In some embodiments, the antibody competitively inhibits binding to the epitope by at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50%.

Antibodies of the present invention may function through different mechanisms. In some embodiments, antibodies have multiple therapeutic functions, including, for example, antigen-binding, the induction of inhibition of VCAM-1 complex formation and transmigration of leukocytes across activated endothelial cells.

In some embodiments, antibodies of the present invention may act as antagonists of the polypeptides of the sixth immunoglobulin-like domain of VCAM-1. For example, in some embodiments the present invention provides antibodies which disrupt the receptor/ligand interactions with the polypeptides of the invention either partially or fully. In some embodiments antibodies of the present invention bind an epitope disclosed herein, or a portion thereof. In some embodiments, antibodies are provided that modulate ligand activity or receptor activity by at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50% compared to the activity in the absence of the antibody.

The antibodies of the present invention may be used either alone or in combination with other compositions. The antibodies may further be recombinantly fused to a heterologous polypeptide at the N- or C-terminus or chemically conjugated (including covalently and non- covalently conjugations) to polypeptides or other compositions. For example, antibodies of the present invention may be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs,- radionuclides, or toxins.

Monoclonal antibodies can be prepared using the method of Kohler et al. (1975) Nature 256:495-496, or a modification thereof. Typically, a mouse is immunized with a solution containing an antigen. Immunization can be performed by mixing or emulsifying the antigen-containing solution in saline, preferably in an adjuvant such as Freund's complete adjuvant, and injecting the mixture or emulsion parenterally. Any method of immunization known in the art may be used to obtain the monoclonal antibodies of the invention. After immunization of the animal, the spleen (and optionally, several large lymph nodes) are removed and dissociated into single cells. The spleen cells may be screened by applying a cell suspension to a plate or well coated with the antigen of interest. The B cells expressing membrane bound immunoglobulin specific for the antigen bind to the plate and are not rinsed away. Resulting B cells, or all dissociated spleen cells, are then induced to fuse with myeloma cells to form hybridomas, and are cultured in a selective medium. The resulting cells are plated by serial or limiting dilution and are assayed for the production of antibodies that specifically bind the antigen of interest (and that do not bind to unrelated antigens). The selected monoclonal antibody (mAb)-secreting hybridomas are then cultured either in vitro (e.g., in tissue culture bottles or hollow fiber reactors), or in vivo (as ascites in mice). As an alternative to the use of hybridomas for expression, antibodies can be produced in a cell line such as a CHO or myeloma cell lines, as disclosed in U.S. Patent Nos. 5,545,403; 5,545,405; and 5,998,144; each incorporated herein by reference. Briefly the cell line is transfected with vectors capable of expressing a light chain and a heavy chain, respectively. By transfecting the two proteins on separate vectors, chimeric antibodies can be produced. Immunol. 147:8; Banchereau et al. (1991) Clin. Immunol. Spectrum 3:8; and Banchereau et al. (1991) Science 251 :70; all of which are herein incorporated by reference.

Antibodies of the present invention may be administered to a subject via in vivo therapeutic antibody gene transfer as discussed by Fang et al. (2005), Nat. Biotechnol. 23, 584-590. For example recombinant vectors can be generated to deliver a multicistronic expression cassette comprising a peptide that mediates enzyme independent, cotranslational self cleavage of polypeptides placed between MAb heavy and light chain encoding sequences. Expression leads to stochiometric amounts of both MAb chains.

Fragments of the antibodies are suitable for use in the methods of the invention so long as they retain the desired affinity of the full-length antibody. Thus, a fragment of an anti-the sixth immunoglobulin-like domain of VCAM-1 antibody will retain the ability to bind to the sixth immunoglobulin-like domain of VCAM-1. Such fragments are characterized by properties similar to the corresponding full-length anti-the sixth immunoglobulin-like domain of VCAM-1 antibody, that is, the fragments will specifically bind a human the sixth immunoglobulin-like domain of VCAM-1 antigen expressed on the surface of a human cell.

In some embodiments, the antibodies bind to one or more epitopes in the sixth immunoglobulin-like domain of VCAM-1. In some embodiments, the antibodies modulate biological activities related sixth immunoglobulin-like domain of VCAM-1. In some embodiments the antibodies inhibit the transmigration of leukocyte across endothelial cell.

Methods of predicting other potential epitopes to which an antibody of the invention can bind are well-known to those of skill in the art and include without limitation, Kyte-Doolittle Analysis (Kyte, J. and Dolittle, R.F., J. MoI. Biol. (1982) 157:105-132), Hopp and Woods Analysis (Hopp, T.P. and Woods, K.R., Proc. Natl. Acad. Sci. USA (1981) 78:3824-3828; Hopp, TJ. and Woods, K.R., MoI. Immunol. (1983) 20:483-489; Hopp, TJ., J. Immunol. Methods (1986) 88:1-18.), Jameson- Wolf Analysis (Jameson, B.A. and Wolf, H., Comput. Appl. Biosci. (1988) 4:181-186.), and Emini Analysis (Emini, E.A., Schlief, W.A., Colonno, RJ. and Wimmer, E., Virology (1985) 140:13-20.).

Antibodies are defined to be "specifically binding" if: 1) they exhibit a threshold level of binding activity, and/or 2) they do not significantly cross-react with known related polypeptide molecules. The binding affinity of an antibody can be readily determined by one of ordinary skill in the art, for example, by Scatchard analysis (Scatchard, Ann. NY Acad. Sci. 51: 660-672, 1949).

In an embodiment of the present invention, Anti-VCAM-1 mAb was isolated using phage display technology from VCAM-1 specific rabbit/human chimeric antibody library. In order to make a VCAM-1 specific rabbit/human chimeric antibody library, we first immunized rabbits with purified recombinant hVCAM-1. Enzyme immunoassay of rabbit sera collected throughout the immunization courses revealed that all rabbits had elevated antibody titers to the antigen (data not shown). After the fourth booster injection, total RNAs were individually isolated from spleen or bone marrow of the immunized rabbits and subjected to cDNA synthesis. Using three steps of PCR, VCAM-1 specific rabbit/human chimeric Fab library containing rabbit variable regions and human constant regions was generated and cloned into phagemid vector pComb3X, yielding a complexicity of 5.7 x 10⁹ independent transformants.

After six rounds of biopanning on immobilized mVCAM-1-Fc using phage display technology, twenty clones were randomly selected, rescued by infection of helper phage, and tested for their reactivity to both hVCAM-1 and mVCAM-1 in phage enzyme immunoassay. Selected clone showing strong reactivity to hVCAM-1 and mVCAM-1 was subsequently analyzed by DNA sequencing and the nucleotide sequences were converted to amino acid sequences using translator program offered by the JustBio webpage (http://www.justbio.com).

Anti-VCAM-1 mAb had strong affinity and broad cross-species relativity to human and mouse VCAM-1. Anti-VCAM-1 mAb specifically recognized the sixth IgG-like domain of VCAM-1 - To identify an epitope region for the anti-VCAM-1 mAb, we first generated Fc fusions of wild-type and C-terminal serial deletion mutants of human and mouse VCAM-1 extracellular domains (Fig. 2A). Then, following overexpression in HEK293F and affinity purification with protein A sepharose, the equal amounts of purified Fc fusion proteins were subjected to immunoblot analysis for eiptope mapping. Here, anti-human Fc-HRP was used to detect the equal quantity of each Fc fusion proteins. As shown Fig. 2B and 2C, this mAb specifically recognized the sixth IgG-like domain of hVCAM-1 and mVCAM-1 respectively.

As used herein, the term "oligonucleotide" refers to a series of linked nucleotide residues. Oligonucleotides include without limitation, antisense and siRNA oligonucleotides. Oligonucleotides comprise portions of a DNA sequence and have at least about 10 nucleotides and as many as about 500 nucleotides. In some embodiments oligonucleotides comprise from about 10 nucleotides to about 50 nucleotides, from about 15 nucleotides to about 30 nucleotides, and from about 20 nucleotides to about 25 nucleotides. Oligonucleotides may be chemically synthesized and can also be used as probes. In some embodiments oligonucleotides are single stranded. In some embodiments oligonucleotides comprise at least one portion which is double stranded. In some embodiments the oligonucleotides are antisense oligonucleotides. In some embodiments the oligonucleotides are RNAi oligonucleotides, siRNAs or shRNAs.

As used herein, the term "antisense oligonucleotide" refers to an unmodified or modified nucleic acid having a nucleotide sequence complementary to polynucleotide sequence of a sixth immunoglobulin-like domain of VCAM-1, where the antisense polynucleotide is capable of hybridizing to polynucleotide sequence of a sixth immunoglobulin-like domain of VCAM-1. Of particular interest are antisense polynucleotides capable of inhibiting transcription and/or translation of polypeptide f a sixth immunoglobulin-like domain of VCAM-1 encoding polynucleotide either in vitro or in vivo.

As used herein, the terms "siRNA oligonucleotides", "RNAi oligonucleotides", "short interfering RNA", or "siRNA" are used interchangeably and refer to oligonucleotides that work through post-transcriptional gene silencing, also known as RNA interference (RNAi). The terms refer to a double stranded nucleic acid molecule capable of RNA interference "RNAi", (see Kreutzer et al., WO 00/44895; Zernicka-Goetz et al. WO 01/36646; Fire, WO 99/32619; Mello and Fire, WO 01/29058). SiRNA molecules are generally RNA molecules but further encompass chemically modified nucleotides and non-nucleotides. SiRNA gene-targeting experiments have been carried out by transient siRNA transfer into cells (achieved by such classic methods as liposome-mediated transfection, electroporation, or microinjection). Molecules of siRNA are 21- to 23-nucleotide RNAs, with characteristic 2- to 3-nucleotide 3'-overhanging ends resembling the RNase III processing products of long double-stranded RNAs (dsRNAs) that normally initiate RNAi. Effective exploitation of the siRNA pathway to mediate gene silencing depends, in part, on efficient methods of intracellular delivery of siRNA. siRNA molecules tend to be short-lived in the cell, not readily deliverable to cell types that are difficult to transfect and relatively expensive to produce via chemical syntheses. (Jacks et al., (2005) Biotechniques 39: 215-224; Bernards et al., (2006) Nature Methods 3: 701-706) One method for efficient intracellular delivery of siRNA is the use of short hairpin RNAs, or "shRNAs". shRNAs are single stranded RNA molecules that include two complementary sequences joined by a non-complementary region. In vivo, the complementary sequences anneal to create a double-stranded helix with an unpaired loop at one end. The resulting lollypop-shaped shaped structure is called a stem loop and can be recognized by the RNAi machinery and processed intracellularly into short duplex RNAs having siRNA-like properties.

shRNA can be synthesized in a cell by transcription from a DNA template that has been inserted into a appropriate vector. Useful shRNAs are typically 50-70 nucleotides in length, with two complementary sequences of 19-29 nucleotides separated by a 5-10 nucleotide loop. shRNA construction is generally effected by one of three methods: annealing of complementary oligonucleotides; promoter-based polymerase chain reaction (PCR); or primer extension. Many vector systems employ RNA Pol IH promoters; Pol Ill- mediated transcription is advantageous because it initiates at a well-defined start-site, produces a non-poly (A) containing transcript and Pol in promoters are active in all cell types. (Brummelkamp et al., (2002) Science 296: 550-553; Mclntyre, G. and Fanning, G. (2006) BMC Biotechnology 6: 1-8)

shRNA-encoding vector systems provide a renewable intracellular source of gene-silencing reagents that can mediate persistent gene silencing after stable integration of the vector into the host genome. Moreover, the shRNA cassette can be readily inserted into retroviral, lentiviral or adenoviral vectors to facility delivery of shRNA into a broad range of cell types, -including nondividing primary cultures. Regulatable versions of shRNA vectors are particularly useful for genetic screens.

Preferably, it is also possible to inhibit activity of the sixth immunoglobulin-like domain of VCAM-1 in combination with the modulators of the sixth immunoglobulin-like domain of VCAM-1. As used herein, the terms "in combination with" or "in conjunction with" refer to administration of the modulators of the sixth immunoglobulin-like domain of VCAM-1 with other therapeutic regimens.

The present invention further provides methods of inhibiting VCAM-1 mediated-diseases in a patient diagnosed or suspected of having a VCAM-1 mediated-diseases. More particularly, the present invention provides a method of preventing or treating VCAM-1 mediated-disease by inhibiting activity of the sixth immunoglobulin-like domain of VCAM-1. The methods may comprise administering a therapeutically effective amount of one or more modulators of the sixth immunoglobulin-like domain of VCAM-1 to the patient. The present invention also provides methods for inhibiting the transmigration of leukocyte across activated endothelial cell in a patient comprising administering a therapeutically effective amount of a modulator of the sixth immunoglobulin-like domain of VCAM-1 to said patient. Suitable assays for measuring transmigration of leukocyte across activated endothelial cell are known to those skilled in the art and are set forth supra and infra.

The methods comprise determining if the patient is a candidate for therapy of VCAM-1 mediated-diseases and administering a therapeutically effective amount of one or more modulators of the sixth immunoglobulin-like domain of VCAM-1 to the patient if the patient is a candidate for therapy of VCAM-1 mediated-diseases.

The terms "patient" or "subject" are used interchangeably and refer to any subject for whom diagnosis, treatment, or therapy is desired, particularly humans. Other subjects may include cattle, dogs, cats, guinea pigs, rabbits, rats, mice, horses, and the like. In some preferred embodiments the subject is a human.

The present invention further provides methods including other active ingredients in combination with the modulators of the sixth immunoglobulin-like domain of VCAM-1 of the present invention. In some embodiments, the methods further comprise administering one or more conventional VCAM-1 mediated-diseases therapeutics to the patient. In some embodiments the methods of the present invention further comprise treating the patient with one or more of chemotherapy, radiation therapy or surgery. The present invention also provides methods and compositions for the treatment, inhibition, and management of VCAM-1 mediated-diseases or disease that has become partially or completely refractory to current or standard VCAM-1 mediated-diseases treatment, such as surgery, chemotherapy, radiation therapy, hormonal therapy, and biological therapy.

A therapeutically effective amount of the modulating compound can be determined empirically, according to procedures well known to medicinal chemists, and will depend, inter alia, on the age of the patient, severity of the condition, and on the ultimate pharmaceutical formulation desired. Administration of the modulators of the present invention can be carried out, for example, by inhalation or suppository or to mucosal tissue such as by lavage to vaginal, rectal, urethral, buccal and sublingual tissue, orally, topically, intranasally, intraperitoneally, parenterally, intravenously, intralymphatically, intratumorly, intramuscularly, interstitially, intra-arterially, subcutaneously, intraoccularly, intrasynovial, transepithelial, and transdermally. In some embodiments, the inhibitors are administered by lavage, orally or inter-arterially. Other suitable methods of introduction can also include rechargeable or biodegradable devices and slow or sustained release polymeric devices. As discussed above, the therapeutic compositions of this invention can also be administered as part of a combinatorial therapy with other known anti-VCAM-1 mediated-diseases agents or other known anti-bone disease treatment regimen.

The present invention provides methods for treating and/or preventing VCAM-1 mediated-diseases or symptoms of VCAM-1 mediated-diseases in a subject.

Prevention or treatment of VCAM-1 mediated disease can be accomplished by inhibiting activity of the sixth immunoglobulin-like domain of VCAM-1 to an effective extent of inhibiting the transmigration of leukocyte across endothelial cell, and more preferably, it is not associated with the binding of leukocyte to endothelial cell. For preventing or treating VCAM-1 mediated disease, one or more modulators of the sixth immunoglobulin-like domain of VCAM-1 in a therapeutically effective amount can be administered to a patient. And, modulators of the sixth immunoglobulin-like domain of VCAM-1 can be administered to a patient using a pharmaceutically acceptable carrier. Also, the method of treating VCAM-1 mediated disease described in this invention can be administered in combination or alternation with a second biologically active agent to increase its effectiveness against the target disorder.

The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, such as antibodies or a polypeptide, genes, and other therapeutic agents. The term refers to any pharmaceutical carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which can be administered without undue toxicity. Suitable carriers can be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates and inactive virus particles. Such carriers are well known to those of ordinary skill in the art. Pharmaceutically acceptable carriers in therapeutic compositions can include liquids such as water, saline, glycerol and ethanol. Auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, can also be present in such vehicles.

“VCAM-1 mediated disease” comprises all disease mediated by VCAM-1. As VCAM-1 mediated diseases, there care cancers, allergic responses, atherosclerosis, cardiovascular diseases, HIV (human immunodeficiency virus, AIDS) disease, arthritis, pneumonia, hypercholesterolemina, sepsis, dermatitis, psoriasis, Crohn's disease, cystic fibrosis, post transplantation late and chronic solid organ rejection, cell or islet transplantation rejection, multiple sclerosis, systemic lupus erythematosis, Graves' disease, thrombotic disease, inflammatory bowel diseases, autoimmune diabetes, diabetic retinopathy, rhinitis, ischemia- reperfusion injury, post-angioplasty restenosis, osteomyelitis, cold, influenza virus disease, chronic obstructive pulmonary disease (COPD), glomerulonephritis, Graves disease, gastrointestinal allergies, sickle cell disease, and conjunctivitis, but will not limited thereto.

Above cancers include beast cancer, brain cancer, lung cancer, leukemia, liver cancer, Non-hodkin’s lymphoma, ovarian cancer, stomach cancer, rectal cancer, colon cancer, pancreatic cancer and kidney cancer, but will not limited thereto.

Above allergic responses include asthma, atopy, eczema, rhinitis and anaphylaxis, but will not limited thereto.

Above cardiovascular diseases include coronary artery disease, heart attack and stroke, but will not limited thereto.

Nontlimiting examples of arthritis include rheumatoid (such as soft-tissue rheumatism and non-articular rheumatism, fibromyalgia, fibrositis, muscular rheumatism, myofascil pain, humeral epicondylitis, frozen shoulder, Tietze's syndrome, fascitis, tendinitis, tenosynovitis, bursitis), juvenile chronic, spondyloarthropaties (ankylosing spondylitis), osteoarthritis, hyperuricemia and arthritis associated with acute gout, chronic gout and systemic lupus erythematosus.

Human endothelial disorders mediated by VCAM-1 include psoriasis, eczematous dermatitis, Kaposi's sarcoma, as well as proliferative disorders of smooth muscle cells.

In yet another embodiment, the method disclosed herein can be selected to treat anti-inflammatory conditions that are mediated by mononuclear leucocytes.

In one embodiment, the methods of the present invention are selected for the prevention or treatment of tissue or organ transplant rejection. Treatment and prevention of organ or tissue transplant rejection includes, but are not limited to treatment of recipients of heart, lung, combined heart-lung, liver, kidney, pancreatic, skin, spleen, small bowel, or corneal transplants. The method can also be used in the prevention or treatment of graft- versus-host disease, such as sometimes occurs following bone marrow transplantation.

In an alternative embodiment, the method described herein are useful in both the primary and adjunctive medical treatment of cardiovascular disease. The method is used in primary treatment of, for example, coronary disease states including atherosclerosis, post-angioplasty restenosis, coronary artery diseases and angina. The method can be administered to treat small vessel disease that is not treatable by surgery or angioplasty, or other vessel disease in which surgery is not an option. The method can also be used to stabilize patients prior to revascularization therapy.

In an embodiment of the present invention, the sixth IgG-like domain of VCAM-1 could be a new molecular target for alleviating mouse allogeneic transplantation rejection (Fig. 5).

Where activity of the sixth immunoglobulin-like domain of VCAM-1 has been inhibited using modulators of the sixth immunoglobulin-like domain of VCAM-1 after having made mouse islet allograft models, a result could be obtained that no plantation rejection has been shown and blood sugar amount has gradually recovered to a normal state.

As another aspect, the present invention relates to a method for screening VCAM-1 mediated-diseases treatment or prevention materials and a kit for the same, the method comprises the steps of: (1)contacting a patient of VCAM-1 mediated-diseases patient with a sample; (2)detecting increase or decrease of activity of the sixth immunoglobulin-like domain of vascular cell adhesion molecule-1 (VCAM-1) expressed on activated endothelial cell; and (3)determining the sample that inhibits activity as a VCAM-1 mediated-diseases treatment or prevention materials.

As used herein, the term "sample" refers to biological material from a patient The sample assayed by the present invention is not limited to any particular type. Samples include, as non-limiting examples, single cells, multiple cells, tissues, tumors, biological fluids, biological molecules, or supematants or extracts of any of the foregoing. Examples include tissue removed for biopsy, tissue removed during resection, blood, urine, lymph tissue, lymph fluid, cerebrospinal fluid, mucous, and stool samples. The sample used will vary based on the assay format, the detection method and the nature of the tumors, tissues, cells or extracts to be assayed. Methods for preparing samples are weli known in the art and can be readily adapted in order to obtain a sample that is compatible with the method utilized.

As used herein, the term "contacting" means bringing together, either directly or indirectly, one molecule into physical proximity to a second molecule. The molecule can be in any number of buffers, salts, solutions, etc. "Contacting" includes, for example, placing a polynucleotide into a beaker, microtiter plate, cell culture flask, or a microarray, or the like, which contains a nucleic acid molecule. Contacting also includes, for example, placing an antibody into a beaker, microtiter plate, cell culture flask, or microarray, or the like, which contains a polypeptide. Contacting may take place in vivo, ex vivo, or in vitro.

As used herein, the term "detecting" means to establish, discover, or ascertain evidence of an activity (for example, gene expression) or biomolecule (for example, a polypeptide). As described in the present invention, the evidence of inhibiting activity can be measured using various method known in this art.

All of the references cited herein are incorporated by reference in their entirety. Also, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention specifically described herein. Such equivalents are intended to be encompassed in the scope of the claims.

According to the present invention, it is expected that it will be possible not only to screen materials that can inhibit the transmigration of leukocyte across endothelial cell without affecting the binding of leukocyte to endothelial cell, but also to effectively prevent or treat VCAM-1 mediated-diseases.

The present invention will become more fully understood from the detailed description given herein below, and the accompanying drawings which are given by way of illustration only, and thus are not limitative of the present invention, and wherein;

Figure 1 shows Anti-VCAM-1 recognizes the domain 6 and 7 of hVCAM-1 and mVCAM-1.

Fig. 1a: the full-length extracellular domain of human and mouse VCAM-1 and its C-terminal deletion mutants constructed according to their domains were prepared as a Fe-fusion proteins.

Fig. 1b: The same amount of the full-length and C-terminal deletion mutants of hVCAM-1 as indicated were loaded onto a gel and subjected to immunoblot analysis using anti-VCAM-1 Fab/anti-human Fab-HRP (upper panel) or anti-human Fc-HRP (lower panel) respectively.

Fig. 1c: The same amount of the full-length and a C-terminal deletion mutant of mVCAM-1 as indicated were loaded onto a gel and subjected to immunoblot analysis using anti-VCAM-1 Fab/anti-human Fab-HRP (upper panel) or anti-human Fc-HRP (lower panel) respectively.

Figure 2 shows Anti-VCAM-1 Fab has an ability to inhibit the transmigration of leukocytes across activated HUVEC. 2x10⁵ of HUVEC was plated on the upper part of transwells. Then, after pre-treating anti-VCAM-1 Fab for 1 hr, U937 was added to the upper part of the transwell and simultaneously, 50 ng/ml of recombinant hSDF-1a was also added to the lower part of the transwell. After 16 hrs, the migrated cells were counted using light microscopy. These results shown represent the means ± S.D. obtained from experiments performed in triplicate.

Figure 3 shows Anti-VCAM-1 Fab little blocks the interaction between leukocytes and activated HAEC and HUVEC - HAEC and HUVEC cultured in the absence (dotted line) or presence (solid line) of hTNF<;x were pre-treated with anti-VCAM-1 Fab for 1 hr as indicated. Then, by incubating CSFE-Iabeled U937 cells for 1 hr, the bound cells were measured using flow cytometry.

Figure 4 shows Anti-VCAM-1 Fab little blocks the interaction between leukocytes and VCAM-1 overexpressing HEK 293 cell lines.

Fig. 4a: Vector-, hVCAM-1-, and mVCAM-1-overexpressing HEK 293 cell lines were subjected to flow cytometry in the absence (dot line) or presence of anti-CAM-1 Fab (solid line).

Fig. 4b: Vector-, hVCAM-1-, and mVCAM-1-overexpressing HEK 293 cell lines were incubated with CSFE-Iabeled U937 or Raw264.7 cell lines for 1 hr as indicated. Then, the bound cells were detected using flow cytomety.

Fig. 4c: The bound leukocytes to vector-(open rectangle), hVCAM-1-(gray rectangle), and mVCAM-1-(black rectangle) overexpressing HEK 293 cell lines were depicted as a graph bar.

These results shown represent the means ± S.D. obtained from experiments performed in triplicate.

Figure 5 showas Anti-VCAM-1 Fab has an ability to inhibit VCAM-1 clustering on HUVEC - Before VCAM-1 cross-linking, the HUVEC plated on cover glass were incubated in the absence or presence of anti-VCAM-1 Fab as indicated. The cross-linking was induced by incubating Alexa Fluor 488-labeled anti-rabbit polyclonal antibody. Then, the signals were detected using confocal microscopy. Here, hoechst and F-actin were stained for detecting individual cells. Arrowhead means the cells showing VCAM-1 clustering.

Figure 6 showas Anti-VCAM-1 Fab specifically inhibit downstream signaling of

VCAM-1 (Rac1-ROS-p38 MAPK) on HUVEC - Before VCAM-1 cross-linking, HUVEC was incubated in the absence or presence of anti-VCAM-1 Fab as indicated.

Fig. 6a: 0.7 mg of each of the cell extracts were then incubated with GST or GST-PBD to detect active form of rac1 followed by immunoblot analysis with anti-rac1 monoclonal antibody. Here, 20 I-lg of each of samples were also loaded to show the equal amount of protein quantity.

Fig. 6b: At the same time of VCAM-1 cross-linking, DCF-DA fluorescent dye was simultaneously added to HUVEC and the changes of fluorescent intensity was measured using flow cytometry.

Fig. 6c: The cell extracts were loaded onto a gel followed by immunoblot analysis with anti-phospho p38 MAPK antibody.

Figure 7 shows Anti-VCAM-1 Fab could alleviate IBMIR in mouse islet allograft-The protocol of i.p. injection of a-VCAM-1 Fab in EG was set in combination of islet transplantation. 1.25 mg of a-VCAM-1 Fab prepared in 88.6ul buffer was injected Lp. two times per day regularly from 0 day post islet transplantation to 9 days post islet transplantation. The BGL of H-2b male recipient mice could not be controlled with 300 lEO graft of H-2d male donor (blue bar) in contrast, the BGL of H-2b male recipient mice could be maintained normal from the early period of islet engraftment continuously during 25 days post islet transplantation with 300 lEO graft of H-2d male donor in EG (red bar).

Figure 8 shows Anti-VCAM-1 mAb had broad cross-species reactivity to human and mouse VCAM-1.

Fig. 8a: After pre-incubation with the indicated amounts of BSA (negative control) and recombinant hVCAM-1 and mVCAM-1 onto 96 well plates, ELISA was performed with purified irrelevant Fab (□) or anti-VCAM-1 Fab (■).

Fig. 8b: The indicated amounts of hVCAM-1 and mVCAM-1 were loaded onto a polyacrylamide gel followed by immunoblot analysis with anti-VCAM-1 Fab.

Fig. 8c: HAEC, HUVEC, and MVEC were cultured in the absence (dotted line) or presence (solid line) of hTNFα or mTNFα. Then, flow cytometry was performed with anti-VCAM-1 Fab.

These results are a representative of three separate experiments.

Figure 9 shows Anti-VCAM-1 mAb recognized the sixth IgG-like domain of hVCAM-1 and mVCAM-1.

Fig. 9a: The indicated wild-type and C-terminal serial domain deletion mutants of hVCAM-1 and mVCAM-1 extracellular domain were constructed and prepared as Fc-fusion proteins.

Fig. 9b: The same amount of purified wild-type and C-terminal serial domain deletion mutants of hVCAM-1 were loaded onto a polyacrylamide gel and subjected to immunoblot analysis using anti-VCAM-1 Fab/anti-human Fab-HRP (upper panel) or anti-human Fc-HRP (lower panel) respectively.

Fig. 9c: The same amount of purified wild-type and C-terminal serial domain deletion mutants of mVCAM-1 were loaded onto a polyacrylamide gel and subjected to immunoblot analysis using anti-VCAM-1 Fab/anti-human Fab-HRP (lower panel) or anti-human Fc-HRP (upper panel) respectively.

These results were a representative of three separate experiments.

Figure 10 shows Anti-VCAM-1 mAb specifically inhibited the transmigration of U937 cells across activated HUVECs. 2x10⁵ of HUVEC plated on the upper part of transwells was cultured in the absence or presence of hTNFα for 1 day. After pre-incubated with anti-VCAM-1 Fab for 1 hr, U937 cells were added to the upper part of the transwell and simultaneously, 50 ng/ml of recombinant hSDF-1α was also added to the lower part of the transwell. The migrated cells were counted using light microscopy.

These results shown represent the means ± S.D. obtained from experiments performed in triplicate.

Figure 11 shows Anti-VCAM-1 mAb little affect the binding of VCAM-1 to α4β1 integrin.

Fig. 11a: Vector- or hVCAM-1-overexpressing HEK293 cell lines were subjected to flow cytometry in the absence (dotted line) or presence (solid line) of anti-VCAM-1 Fab.

Fig. 11b: HAEC (white), HUVEC (gray), vector- or hVCAM-1-overexpressing 293 cell lines (black) were incubated in the absence or presence of anti-VCAM-1 Fab for 1 hr. Following the incubation of CSFE-labeled U937 cells for 1 hr, the bound cells were measured using flow cytometry.

Fig. 11c: The mean fluorescence intensity of bound CSFE-labeled U937 cells is depicted as a vertical bar.

Data are shown as the means ±S.D. obtained from a representative of three separate experiments performed in duplicate.

Figure 12 shows Anti-VCAM-1 mAb significantly alleviated mouse islet allogeneic transplantation rejection.

Fig. 12a: 250 IEQ of isolated mouse islets from BALB/c (donor) was grafted to C57BL/6j (recipient) rendered Diabetes Mellitus by streptozotocin. Then, PBS or 100 ㎍ of purified anti-VCAM-1 full-IgG was injected to the recipient via intraperitoneal injection everyday up to 7 days.

Fig. 12b: Blood glucose level was determined with portable glucometer from obtained from the snipped tail of PBS (closed circle) or anti-VCAM-1 full-IgG (open circle)-treated groups.

A better understanding of the present invention may be obtained through the following examples which are set forth to illustrate, but are not to be construed as the limit of the present invention.

EXAMPLE 1

Cell culture

Vector-, human VCAM-1(hVCAM-1)-, or mouse VCAM1(hVCAM-1 )-transfected human embryonic kidney (HEK) 293 or HEK293T cell lines was maintained in Dulbecco's modified Eagle's medium (DMEM;Invitrogen) supplemented with 10% (v/v) fetal bovine serum (FBS) (Invitrogen) and 1% (v/v) penicillin/streptomycin (Invitrogen). Human umbilical vein endothelial cells (HUVECs) and human aortic endotheluial cells (HAEC) were maintained in endothelial growth media-2 (EGM-2) followed by manufacturer's instruction (Lonza). Mouse vascular endothelial cell (MVEC) lines kindly donated by Dr. Saito (Tsurumi University, Tsurumi, Japan) were in Medium 199 supplemented with 5% (v/v) FBS, 10 ㎍/ml insulin, 2.4 ㎍/ml hydrocortisone, and 1% (v/v) penicillin/streptomycin (Invitrogen). U937 human monocytic and Raw 264.7 mouse macrophage cell lines were in RPMI supplemented with 10%(v/v) fetal bovine serum (FBS) (Invitrogen) and 1% (v/v) penicillin/streptomycin (Invitrogen). All cells were cultured at 37℃ in a humidified C02-controlled (5%) incubator.

Flow cytometry

Flow cytometry was performed as described previously(international immunology). Briefly, the endothelial cells cultured in the absence or presence of indicated stimuli were incubated with 10 ㎍/ml of anti-VCAM-1Fab in blocking buffer containing 1% (w/v) BSA in PBS at 37℃ for 1 hr. After three times washings with blocking buffer, the cells were then incubated with alexa flour 488-labeled anti-human Fab antibody (1:100) (Invitrogen) at 37℃ for 1 hr. The final pellets were subjected to flow cytometry (Beckmann Coulter, Miami, Florida, USA).

Cell adhesion and neutralization assay

Leukocyte adhesion assays were performed with minor modification as described. Briefly, 3 x 10⁵ cells of endothelial cells plated on 6 well dishes were stimulated by indicated stimuli for 1 day. Following CFSE labeling with U937 promonocytic leukocytes, the labeled cells were incubated with activated endothelial cells for 1 hr at 37℃ and then unbound cells were washed 5 times with 1x PBS containing 0.2 mM CaCl₂ and 0.1 mM MgCl₂. The final cells were trypsinized and then subjected to flow cytometry. For neutralizing assay, the activated endothelial cells were incubated with 50 ㎍/ml of anti-VCAM-1 Fab for 1 hr at 37℃ in prior to the addition of CFSE labeled U937. Following procedures are the same to above procedures.

Transendothelial cell migration assay

A modified Boyden chamber assay was used to study the migration of U937 cells as previously described O.2 x 10⁵ of HAEC, HUVEC, and MVEC were added to transwell polycarbonate membranes with 3 ㎛ pores (Costar, Corning, Acton, MA) overnight at 37℃, followed by treating the cells with 20 ng/ml of hTNFa or mTNFa respectively.

After I day incubation, U937 cells (2x10⁵ cells/well) were resuspended in U937 medium and placed in the upper chamber. Simultaneously, about 1 ml of U937 cell medium containing 25 ng/mL SDF-1a (R&D Systems) was placed in the lower chamber. After 4 h of incubation, cells in the lower chamber were collected and counted under a light microscope. To investigate the effect of anti-VCAM-1 Fab on transmigration of U937 cells, 50 or 100 ㎍/ml of anti-VCAM-1 Fab was treated to the inserts in prior to adding U937 cells for 1 hr at 37℃.

G eneration of deletion mutants of human and mouse VCAM-1-Fc

Cterminal deletion mutants of the extracellular domains of hVCAM-1 and mVCAM-1 were generated by PCR using following primers: all forward primer of hVCAM-1 and the deletion mutants was used for 5'-GGCCCAGGCGGCCATGCCTGGGAAGATGGTCG-3'. hVCAM-1-R; 5'- GGCCCCACCGGCCCCCTCAGGAGAAAAATAGTCTTTGTT-3', hVCAM-1 (L'1590-698)-R; 5'-GGCCCCACCGGCCCCTCTGCTTCTTCCAGCCTGGT-3', hVCAM-1 (~511-698); 5'-GGCCCCACCGGCCCCGGCAACATTGACATAAAGTG-3', hVCAM-1 (~407-698); 5'GGCCCCACCGGCCCCTGGATCTCTAGGGAATGAGT-3', hVCAM-1 (~311698); 5'-GGCCCCACCGGCCCC TTTCTCTTGAACAATTAATT-3', hVCAM-1 (~222-698); 5'-GGCCCCACCGGCCCCTGATATGTAGACTTGCAATT-3'. All forward primer of mVCAM-1 and the deletion mutants was used for 5'-GGCCCAGGCGGCCATGCCTGTGAAGATGGTCGCGGTCTTGG-3'. mVCAM1-R; 5'-GGCCCCACCGGCCCC TTCGGGCGAAAAATAGTCCTTGTTATGT-3', mVCAM-1 (~525-698)-R; 5'-GGCCCCACCGGCCCC TAGTATAGGAGAGGGGCTGA-3', The PCR products were digested with Sfi I and and then ligated with modified pcDNA 3.1 vectors (Invitrogen), which contains the hinge region and CH2-CH3 domain of human immunoglobulin G1 and finally produces the Fc fusion protein. The sequences of all deletion mutants of human and mouse VCAM-1 were confirmed by DNA sequencing.

Purification of Fc fusion proteins

HEK 293T cell lines were plated at a density of 5 x 10⁶ cells/dish in 100-mm dishes and transfected with 16 ㎍ of individual cDNAs using TurboFect™ (Fermentas) according to manufacturer's instructions O. After 6 days, the culture media were harvested and incubated respectively with protein A sepharose overnight at 4℃. Following several two times washings with PBS, the final pellets in sample buffer were boiled at 95℃ for 5 min and subjected to immunoblot analysis.

Immunocytochemistry

Immunocytochemistry was performed as as previously described with minor modifications. In brief, coverslips were incubated with 1 ㎍/ml of poly-L-Iysine for 1 hr and then HAEC, HUVEC, or MVEC were grown on coverslips. Next, the cells were incubated in the absence or presence of 50 ㎍/ml of anti-VCAM-1 Fab for 30 min at 37℃. Then, 10 ㎍/ml of anti-VCAM-1 polyclonal antibody was also incubated with the cells for 30 min at 37℃. After VCAM-1 was cross-linked with the alexa flour 488-conjugated anti-rabbit secondary antibodies (1 :200) for the indicated periods, the cells were washed with ice-cold PBS two times and fixed with 3.7% (w/v) paraformaldehyde for 30 min at 37℃. Afterwashing with PBS two times, slides were then examined under a fluorescece microscope (Olympus, Melville, NY).

Rac1 activation

The Rac activity assay was performed as previously described with minor modifications. Briefly, 3 x10⁵ of HUVEC, HAEC, and MVEC cultured were stimulated overnight with hTNFa (20 ng/ml) and incubated for 30 min with anti-VCAM-1 antibodies. After VCAM-1 was cross-linked with the secondary antibody for the indicated periods, cells were washed with ice-cold PBS and subsequently lysed for 10 min on ice in PBS containing 5 mM MgCl₂, 1% TX-100, and 1 mM PMSF with brief sonication. Cleared extracts were incubated for 30 min at 4℃ with glutathione S-transferase-p21-activated kinase(GST-PAK) protein, after which glutathione-Sepharose beads were added to precipitate GTP-bound Rac. Total Iysates and precipitates were analyzed on Western blot with the MAb against Rac1 (BD Pharmingen).

Measurement of ROS generation

3 x10⁵ of HAEC, HUVEC, and MVEC cultured were stimulated overnight with hTNFa (20 ng/ml) and incubated for 30 min with anti-VCAM-1 antibodies. Then, the culture media was changed with HBSS (to avoid the interference of phenol red) containing 2% FBS and then DCF-OA was added (final concentration, 1 I-IM) with or without the secondary antibodies for cross-linking to HAEC, HUVEC, and MVEC respectively. The cells were trypsinized (0.05% trypsin) and then subjected to flow cytometry (Beckmann Coulter, Fullerton, CA).

Immunoblot Analysis

The immunoblot analysis was performed as followings. After assaying with Bradford solution for standardization, proteins were denatured by boiling for 5 min at 95℃ in a Laemmli sample buffer, separated by SDS-PAGE, and transferred to nitrocellulose membranes by electroblotting using the wet transfer system (Amersham Biosciences). After blocking in TTBS buffer (10 mM Tris/HCI, pH 7.5, 150 mM NaCI, and 0.05% Tween 20) containing 5% (w/v) skim milk powder, the membranes were incubated with individual monoclonal or polyclonal antibodies, which was followed by another incubation with anti-mouse or anti-rabbit immunoglobulin G coupled with horseradish peroxidase as required. Detection was performed using an enhanced chemiluminescence kit according to manufacturer instructions.

Detection of phosphor-p38 MAPK

3 x10⁵ of HAEC, HUVEC, and MVEC were grown in 6 well dishes, stimulated overnight with hTNFa (20 ng/ml), and pre-treated with 50 ㎍/ml of anti-VCAM-1 Fab for 30 min. Next, the cells were also treated with 10㎍/ml of anti-VCAM-1 polyclonal antibody, washed and incubated with the cross-linking secondary antibody. At the indicated times, the cells were washed with ice-cold PBS and lysed in PBS containing 1% TX-100 with bried sonication. The samples were subjected to immunoblot analysis as described above. Here, anti-phospho-p38 MAPK antibody (1 :2000 in TTBS) (Cell Signaling Technology Inc) was used for detecting phosphorylated p38 MAPK.

Mice

The C57BL/6j (10 weeks age old, H-2J) male mice and Balb/c male mice (10 weeks age old, H-2d) (which had been purchased from Charles liver Jackson laboratory (Bar Harbor, ME) were used. These mice were housed in the Laboratory Animal Research Center at the Samsung Medical Center following Committee Guidelines. The C57BL/6j mice were divided into two groups:1) The control group (CG) underwent Lp. injection of PBS 88.6ul x2 times per day for 10 days(from 0 days post transplantation to 9 days post transplantation). 2) The experimental group (EG) underwent Lp. injection of anti VCAM-1 Fab 1.25mg prepared in PBS 88.6ul x2 times per day for 10 days(from odays post transplantation to 9 days post transplantation).

Induction of diabetes mellitus of mice and monitering

C57BL/6j mices(10 weeks age old) were rendered diabetic by intraperitoneal administration of 200 mg/kg streptozotocin (STZ; Sigma, St. Louis, MO) freshly dissolved in citrate buffer, and they were transplanted 3 days after STZ administration, only after two consecutive non-fasting blood glucose readings of >250 mg/dl were obtained from whole blood. In detail, diabetes was confirmed by the presence of hyperglycemia, weight loss, and polyuria. Only those mice with non-fasting blood glucose level between 400 mg/dl and 450 mg/dl were used as recipients. Blood glucose was measured on days 0, 1, 2, 3, 4, 5, 6, 7 and weekly until days 25 after transplantation. Blood glucose, determined between 9:00 and 11 :00 A.M. in non-fasting conditions, was obtained from the snipped tail, and measured by a portable glucose meter (L1FESCAN INC., Milpitas, CA).

Isolation of islets of Langerhans

Murine islets were isolated as described previously [Lacy, 1967 #29]. Briefly, animals were killed by cervical dislocation, and the pancreas was exposed and injected with Hanks' balanced salt solution (HBSS; Mediatech, Herndon, VA) containing 0.55 mg/ml collagenase (Roche, Indianapolis, IN) via the common bile duct until distension was achieved. Digestion was performed at 37℃ for 17 min with gentle shaking and terminated by the addition of cold RPMI-10% FCS and 2 mmol/l L-glutamine (GIBCO-BRL, Grand Island, NY). Mechanical disruption of the pancreas was achieved by passages through a metal mesh, and islets were purified on Euro-Ficoll (Sigma, St. Louis, MO) gradients by centrifugation at 900g for 11 min, routinely yielding preparations of >90% purity. Islets were hand-picked, counted and scored for

size. An algorithm was used for the calculation of a 150-lJm-diameter islet equivalent number (IE). Islets were stabilized by culturing in RPMI1640 medium supplemented with 11 mM glucose, 2 mM L-glutamine, 10% FCS, 100 Ulml penicillin, and 100 g/ml streptomycin (complete medium) for 2 hrs before the experiments.

Islet transplantation

Three-hundreds IE(lslet Equivalents) of Balb/c male mice were transplanted under the left kidney capsule of C57BL/6j male mice made diabetic by intraperitoneal injection of 200 mg/kg streptozotocin (STZ) (Sigma, St. Louis, MO) freshly dissolved in citrate buffer (pH 4.5). 300 IE has been chosen as it has been known as our previous unpublished data. Before transplantation, diabetes was confirmed.

<1. RESULT>

I dentification of an epitope region against anti - VCAM -1 Fab

To identifyan epitope region for the anti-VCAM-1 Fab, we first generated C-terminal serial deletion mutants of the extracellular domains of hVCAM-1 and mVCAM-1 as Fc fusion forms as indicated in Fig. 1A. Then, following purification, the purified Fc fusion proteins were subjected to immunoblot analysis using anti-VCAM-1 Fab. Here, anti-human Fc-HRP was used to detect the equal quantity of each Fc fusion proteins. As shown Fig. 1B and 1C, anti-VCAM-1 Fab recognizes the domain 6 and 7 of hVCAM-1 and mVCAM-1 respectively.

Anti - VCAM -1 Fab could specifically inhibit the transmigration of U937 human monocytic leukocytes across activated HUVEC

To check the in vitro efficacy of the antibody, HUVEC were plated on the transwell (Corning), stimulated with hTNFa, and subjected to transendothelial cell migration assay with U937 human monocytic cell lines after pre-treating the cells with anti-VCAM-1 Fab. As shown in Fig. 2, we found that anti-VCAM-1 Fab could almost completely inhibits the transmigration of leukocytes across the activated endothelial cells.

Anti - VCAM -1 Fab little blocks the interaction between leukocytes and activated endothelial cells

It is well-known that the domain 1 and 4 of VCAM-1 plays a key role in the binding of leukocytes to activated endothelial cells by interacting with a4~1 integrin. To test the in vitro efficacy of this antibody on inhibiting both interactions, HAEC and HUVEC stimulated with hTNFa were incubated with CSFE-Iabeled U937 cell lines to activated endothelial cells in the absence or presence of anti-VCAM-1 Fab and followed by flow cytometry. As shown Fig. 3A and B, we found that the antibody little neutralizes the VCAM-1-mediated binding of leukocytes to activated endothelial cells. To further confirm the neutralizing effect of the antibody, we plated the same numbers of vector-, hVCAM-1-, or mVCAM-1-overexpressing HEK 293(Fig. 4A) on 6 well plates, incubated with CSFE-Iabeled U937 or Raw264.7 cell lines, and measured the numbers of the bound leukocytes after the treatment of anti-VCAM-1 Fab using flow cytometry(Fig. 4B and 4C). The result also revealed that the antibody could not inhibit the direct binding of leukocytes to hVCAM-1 and mVCAM-1.

Anti - VCAM -1 Fab has an ability to inhibit VCAM -1 clustering on HUVEC

To identify the mode of action of anti-VCAM-1 Fab, after pre-treating the cells with anti-VCAM-1 Fab as indicated in Fig. 5, anti-VCAM-1 polyclonal antibody and its secondary antibodies were also subsequently treated to induce VCAM-1 clustering, a cellular event occurring after VCAM-1-mediated leukocytes/endothelial cells interaction. As shown in Fig. 5, we found that out anti-VCAM-1 Fab has an ability to specifically inhibit VCAM-1 clustering on HUVEC.

Anti - VCAM -1 Fab inhibits VCAM -1- mediated downstream signaling pathway on activated HUVEC

It has been elucidated that VCAM-1 activation by leukocyte adhesion also lead to the activation of VCAM-1 downstream signaling pathway: rac1 activation, NADPH oxidase activation resulting ROS generation, and p38 MPAK activation (phosphorylation). To confirm whether our anti VCAM-1 Fab could inhibit VCAM-1-mediated downstream signaling pathway, we pre-treated HUVEC stimulated with hTNFa with anti-VCAM-1 Fab followed by VCAM-1 cross-linking.

Then, to check the effect of the antibody on rac1 activation, we incubated the cell extracts with GST or GST-PBD and then subjected to immunoblot anlaysis. As shown in Fig. 6A, we found that our anti-VCAM-1 Fab specifically inhibits the Rac1 activation induced by VCAM-1 cross-linking on HUVEC. To further examine whether this antibody could block ROS generation produced by VCAM-1 activation, after the VCAM-1 cross-linking in the presence of DCF-DA, the changes of fluorescence were measured using flow cytometry. As shown in Fig. 6B, the antibody was found to inhibit ROS generation after VCAM-1 cross-linking on HUVEC. To further check the effect of the antibody on p38 MAPK activation, we monitored the phosphorylation of p38 MAPK after VCAM-1 cross-linking in the absence or presence of anti-VCAM-1 Fab. As shown in Fig. 6C, we found that this antibody could also inhibit p38 MAPK induced by VCAM-1 cross-linking on HUVEC.

Anti - VCAM -1 Fab significantly alleviates IBMIR at a mouse islet allograft model

In order to investigate in vivo efficacy of anti-VCAM-1 Fab, we first set mouse islet allograft models. In detail, 300 IEQ of BALB/c male mice (H-2d) was engrafted under the beneath of left kidney capsule in C57BL/6j (H-2b) male recipient mice rendered diabetes Mellitus by Streptozotocin. Then, we treated the buffer (negative control; blue line) or purified anti-VCAM-1 Fab (red line) via daily twice intraperitoneal (i.p) injection at mouse islet allograft models up to 10 days. As shown in Fig. 7, we found that the graft of 300 lEO of BALB/c male mice (H-2d) could control blood glucose level of C57BL/6j (H-2b) male recipient mice in EG, in contrast that the same graft could not control blood glucose level of C57BL/6j (H-2b) male recipient mice in CG. The blood glucose level was maintained lower than 250 mg/dl in EG within the 7 days post islet transplantation when Immediate blood mediated inflammatory reaction (IBMIR) processes and islet can be lost due to IBMIR. In contrast, the blood glucose level was higher than mg/dl in EG within the 7 days post islet transplantation when Immediate blood mediated inflammatory reaction (IB-MIR) processes and islet can be lost due to IB-MIR. Since 7 days post transplantation, the blood glucose level of EG became to be perfect normal until 25 days post transplantation. The blood glucose level of EG became to be abnormal, higher than 250 mg/dl actually.

The inventors of the present invention tested more deeply to find out the specific domain combined with the VCAM-1 antibody among domains of VCAM-1 so as to show the aforementioned effect, and hereafter it will be described.

EXAMPLE 2

Materials

5, 6-carboxy-fluorescein succinimidyl ester (CFSE), Alexa Fluor-labeled goat anti-rabbit secondary antibody, rhodamine phalloidin, and Hoechst were obtained from Molecular Probes. Horseradish peroxidase (HRP)-conjugated antibodies to mouse or rabbit IgG were purchased from Amersham Biosciences (Uppsala, Sweden). Paraformaldehyde, MPL+TDM+CWS adjuvant, methothrexate, and poly-L-lysine were from Sigma. Human and mouse TNFα (hTNFα) and Labscale TFF System were from Millipore (Bedford, MA, USA). HUVEC, HAEC, and endothelial growth media-2 (EGM-2) bullet kits were from Lonza (Baltimore, MD, USA). Penicillin/streptomycin, fetal bovine serum (FBS), RPMI 1640, Freestyle ^TM 293 expression media, CD OptiCHO^TM complete medium, and Dulbecco's modified Eagle's minimal essential medium (DMEM) were purchased from Invitrogen (Gaithersburg, MD, USA). Recombinant human SDF-1α , human VCAM-1 (hVCAM-1), and Fc chimeras of human and mouse VCAM-1 (mVCAM-1) were from R&D systems (Minneapolis, MN, USA).

Cell culture

Vector- or hVCAM-1-transfected human embryonic kidney (HEK) 293 was maintained in DMEM supplemented with 10% (v/v) FBS and 1% (v/v) penicillin/streptomycin. HEK293F was in Freestyle ^TM 293 expression media supplemented with 0.5% (v/v) penicillin/streptomycin. HUVEC and HAEC were maintained in EGM-2 followed by manufacturer’s instruction. Mouse vascular endothelial cell (MVEC) lines kindly donated by Dr. Saito (Tsurumi University, Tsurumi, Japan) were in Medium 199 supplemented with 5% (v/v) FBS, 10 ㎍/ml insulin, 2.4 ㎍/ml hydrocortisone, and 1% (v/v) penicillin/streptomycin. U937 human monocytic cell lines were in RPMI 1640 supplemented with 10% (v/v) FBS and 1% (v/v) penicillin/streptomycin. HEK293F and the other cells were cultured at 37 ℃ in a humidified CO2-controlled (8% or 5%) incubator respectively.

Construction of antibody library and selection of binders

The protocol was performed as described previously (5). In brief, recombinant hVCAM-1 was injected to two different groups of two New Zealand white rabbits under approval of the Institutional Animal Care and Use Committee of the Seoul National University Hospital. After final booster injection, total RNA was prepared from the spleen and the bone marrow and cDNA was synthesized using SuperScript^TM First-Strand (Invitrogen, USA). Following the construction of a rabbit/human chimeric Fab library, Fab clones were selected from the library, through a total of six rounds of biopanning on 2.5 ㎍ of mVCAM-1-Fc coated-Dynabeads M-270 epoxy (Invitrogen, USA) using phage display technology as described (14). After the last round of panning, phage were produced from single clones grown on output plates and tested for binding to human and mouse VCAM-1-Fc by phage enzyme immunoassay as described (15).

Preparation of anti-VCAM-1 Fab

HB2151 E.coli transformed with phagemid DNA was grown in LB medium containing 50 ㎍/ml carbenicillin. The supernatants collected from E.coli were concentrated to approximately 10 times with Labscale TFF System and then incubated with anti-HA antibody-protein A Sepharose complex. After several washings with ImmunoPure(G) IgG binding buffer (Thermo Fisher Scientific Inc, Rockford, IL), the bound proteins were eluted with ImmunoPure IgG elution buffer (Thermo Fisher Scientific Inc, Rockford, IL) and the fraction was immediately neutralized with 1 M Tris, pH 9.2 to adjust physiological pH. The purity of the proteins was evaluated with Coomassie Brilliant staining. In this study, anti-VCAM-1 Fab provided by Hanwha Petroleum Company was partly used.

Enzyme-linked immunosorbent assay (ELISA)

After pre-incubation of the indicated amounts of antigens overnight at 4 ℃, a microtiter plate was blocked with 3% (w/v) BSA in PBS, incubated with the 10 ㎍/ml of irrelevant Fab and anti-VCAM-1 Fab for 1 hr at 37 ℃, and washed two times with PBS containing 0.05% Tween 20. The extent of antibody bound to the antigens was detected by the application of HRP-conjugated anti-human Fab specific antibody. Optical density was measured at 450 nm by a microtiter plate reader (Labsystems, Barcelona, Spain) after incubation with ultra TMB substrate solution (GenDEPOT, Barker, TX).

Immunoblot Analysis

Immunoblot analysis was performed as described previously (16). Briefly, after measuring protein concentration using the Bradford assay, denatured proteins in a Laemmli sample buffer were separated by SDS-PAGE, and transferred to nitrocellulose membranes by electroblotting using the wet transfer system (Amersham Biosciences). After blocking in TTBS buffer (10 mM Tris/HCl, pH 7.5, 150 mM NaCl, and 0.05% Tween 20) containing 5% (w/v) skim milk powder, the membranes were incubated with anti-human Fc-HRP (1:5,000) or 10 ㎍/ml of anti-VCAM-1 Fab, which was followed by another incubation with anti-human Fab-HRP (1:1,000). Detection was performed using a SuperSignal West Pico Chemiluminescence substrate (Pierce, IL, USA) according to the manufacturer's instructions.

Flow cytometry

Flow cytometry was performed as described previously (5). Briefly, the endothelial cells cultured in the absence or presence of indicated stimuli for 1 day were harvested and incubated with 10 ㎍/ml of anti-VCAM-1 Fab in blocking buffer containing 1% (w/v) BSA and 0.05% (w/v) sodium azide in PBS at 37 ℃ for 1 hr. After three times washings with blocking buffer, the cells were then incubated with FITC-labeled anti-human Fab antibody (1:100) (Jackson Immunoresearch Laboratory Inc, Baltimore, PA) at 37 ℃ for 1 hr. After several washings with PBS, the final pellets were subjected to flow cytometry (Beckmann Coulter, Miami, FL).

Real-time interaction analysis

The kinetic parameters of the interaction among the anti-VCAM-1 Fab, anti-VCAM-1 full-IgG, and VCAM-1 were determined using the BIAcore system X-100 (Biacore AB, Uppsala, Sweden). Briefly, human and mouse VCAM-1-Fc were immobilized on a CM5 dextran sensor chip (Biacore AB) in 10 mM sodium acetate buffer (pH 4.0) at a flow rate of 5 ㎕/min using the amine coupling kit. Anti-VCAM-1 Fab and IgG in HEPES- buffered saline containing 0.005% surfactant P20, 3 mM EDTA, and 0.15 M NaCl were injected over 2 min 30 sec at a flow rate of 30 ㎕/min at 37 ℃ and the surface was regenerated with 1 M NaCl/50 mM NaOH. Biacore X-100 evaluation software version 1.1 was used to fit the data and calculate KD values for the binding of the antibody to antigens.

Epitope mapping

The wild-type (WT) and C-terminal serial domain deletion mutants (hVCAM-1-C1~C5, mVCAM-1-C1~C2) of hVCAM-1 or mVCAM-1 extracellular domain depicted in Fig. 2A were generated by PCR with synthetic primers. The PCR products of wild-type and the serial deletion mutants were digested with Sfi I and then ligated with modified pcDNA 3.1 vectors (Invitrogen, USA), which contains the hinge region and CH2-CH3 domain of human IgG1 and finally produces the Fc fusion protein. The nucleotide sequences of all constructs were confirmed by DNA sequencing. Then, 50 ㎍ of individual DNAs were transfected to 4 x 10⁷ of HEK293F cell lines respectively using 50 ㎍ of polyethylenimine (Polyscience, Warrington, PA). After one week, the culture media were harvested and purified using affinity chromatography with protein A sepharose overnight at 4℃. Following several two times washings with PBS, the final pellets in sample buffer were boiled at 95℃ for 5 min and subjected to immunoblot analysis.

Transendothelial cell migration assay

2 x 10⁵ of HUVEC was added to transwell polycarbonate membranes with 3 ?m pores (Corning Inc, Coring, NY) overnight at 37℃, followed by treating the cells with 20 ng/ml of hTNFα. After 1 day incubation, U937 (2x10⁵ cells/well) human monocytic cell lines were placed in the upper chamber. Simultaneously, about 1 ml of RPMI1640 medium containing 25 ng/ml of human SDF-1α was placed in the lower chamber. After 1 day of incubation, the migrated cells in the lower chamber were collected and counted under a light microscope. To investigate the inhibitory effect of anti-VCAM-1 Fab on leukocyte transmigration, 50 or 100 ㎍/ml of anti-VCAM-1 Fab was treated to the inserts in prior to adding U937 cells for 1 hr at 37 ℃.

Generation of HEK 293 cell line stably overexpressing the full-length of hVCAM-1

The full-length cDNA of hVCAM-1 was ligated into the KpnI or XhoI site of pcDNA3.1(+) vector (Invitrogen). 5 x 10⁴ of HEK293 cell lines was transfected with 4 ㎍ of the expression vector encoding the full-length of hVCAM-1 with 6 ㎕ of TurboFect^TM (Fermentas International, Inc., Burlington, Canada) as manufacturer’s recommendation. Then, after 1 day, the cells were cultured in DMEM supplemented with 10% (v/v) FBS, 1% (v/v) penicillin/streptomycin, and 400 mg/ml of G418 as a selection marker for more than 1 month to select HEK293 cell line stably expressing the full-length of hVCAM-1. The expression of hVCAM-1 on the selected clones were confirmed by flow cytometry.

Cell adhesion and neutralization assay

Leukocyte adhesion assays were performed with minor modification as described (17). Briefly, 3 x 10⁵ cells of vector-transfected (MOCK) or hVCAM-1 overexpressing HEK293 cell lines, HAEC, and HUVEC were plated on 6 well dishes. Human endothelial cells were then stimulated by hTNFα for 1 day. Following CFSE labeling with U937 cells, the labeled cells were incubated with activated endothelial cells or the HEK293 cell lines for 1 hr at 37℃ and then unbound cells were washed 5 times with 1x PBS containing 0.2 mM CaCl₂ and 0.1 mM MgCl₂. The final cells were trypsinized and then subjected to flow cytometry. For neutralizing assay, the activated endothelial cells or the HEK293 cell lines were incubated in the absence or presence of 50 ㎍/ml of anti-VCAM-1 Fab for 1 hr at 37℃ in prior to the addition of CFSE labeled U937 cells. Following procedures are the same to above procedures.

Preparation of anti-VCAM full-IgG

CHO-DG44 cell lines () were transfected with a vector encoding anti-VCAM-1 Full-IgG using electroporation with MicroPulser^TM electroporator (Bio-Rad, USA). After 2 days, 5x 10² of the cells were transferred to 96 well plates and cultured in CD OptiCHO^TM complete medium supplemented with 200 mM L-glutamine, 500 mg/ml G418 (GIBCO-BRL, Grand Island, NY), and 25 nM methothrexate for 19 day. Then, the culture media were subjected to ELISA to select strong clone to express antibody having dual cross species reactivity to hVCAM-1 and mVCAM-1. Through several rounds of selection procedures, the final clones were grown in 1L of VWR Polycarbonate erlenmeyer flask containing CD OptiCHO^TM complete medium supplemented with 500 mg/ml G418 and 200 mM L-glutamine at 37C in a shaking incubator (Infors HT Mini-tron, Bottmingen, Switzerland). Following centrifugation at 6,000 x g for 30min, the supernatant was subjected to affinity chromatography with protein A Sepharose column.

Isolation of mouse pancreatic islets

Murine islets were isolated as described previously (18). Briefly, the mouse pancreas harvested from Balb/c male mice (10 weeks age old, H-2d: donor) (Charles liver Jackson laboratory, Bar Harbor, ME) was exposed and injected using Hanks’ balanced salt solution (HBSS; Mediatech, Herndon, VA) containing 0.8 mg/ml collagenase P (Roche, Indianapolis, IN) via the common bile duct until distension was achieved. The distended pancreas was digested at 37℃ for 12 min with gentle shaking and terminated by the addition of cold RPMI-10% FCS and 2 mM L-glutamine (GIBCO-BRL, Grand Island, NY). The islets achieved by passages through a metal mesh were purified on Euro-Ficoll (Sigma, St. Louis, MO) gradients by centrifugation at 900 x g for 11 min, routinely yielding preparations of >90% purity. Islets were stabilized by culturing RPMI 1640 medium supplemented with 11 mM glucose, 2 mM L-glutamine, 10% FCS, 100 U/ml penicillin, and 100 g/ml streptomycin (complete medium) for 2 hrs before the experiments.

Establishment of mouse islet allograft model

250 Islet Equivalents (IEQ) of Balb/c male mice were transplanted under the left kidney capsule of C57BL/6j male mice (10 weeks age old, H-2b: recipient) made diabetic by intraperitoneal injection (i.p) of 200 mg/kg streptozotocin (STZ) (Sigma, St. Louis, MO) freshly dissolved in citrate buffer (pH 4.5). The C57BL/6j mice were divided into two groups: 1) the control group was treated with 100 ㎕ of PBS via i.p. injection per day for 8 days (from 0 days post transplantation to 7 days post transplantation). 2) The experimental group was treated with of 100 ㎍ of anti-VCAM-1 full-IgG in PBS 100 ㎕ via i.p. injection per day for 7 days (from 0 days post transplantation to 7 days post transplantation). Blood glucose was measured on every day until 31 days after transplantation. Blood glucose in non-fasting conditions was obtained from the snipped tail, and measured by a portable glucometer (Lifescan Inc., Milpitas, CA).

<2. RESULT>

Anti - VCAM -1 mAb was isolated using phage display technology from VCAM -1 specific rabbit / human chimeric antibody library

In order to make a VCAM-1 specific rabbit/human chimeric antibody library, we first immunized rabbits with purified recombinant hVCAM-1. Enzyme immunoassay of rabbit sera collected throughout the immunization courses revealed that all rabbits had elevated antibody titers to the antigen (data not shown). After the fourth booster injection, total RNAs were individually isolated from spleen or bone marrow of the immunized rabbits and subjected to cDNA synthesis. Using three steps of PCR, VCAM-1 specific rabbit/human chimeric Fab library containing rabbit variable regions and human constant regions was generated and cloned into phagemid vector pComb3X, yielding a complexicity of 5.7 x 10⁹ independent transformants.

After six rounds of biopanning on immobilized mVCAM-1-Fc using phage display technology, twenty clones were randomly selected, rescued by infection of helper phage, and tested for their reactivity to both hVCAM-1 and mVCAM-1 in phage enzyme immunoassay. Selected clone showing strong reactivity to hVCAM-1 and mVCAM-1 was subsequently analyzed by DNA sequencing and the nucleotide sequences were converted to amino acid sequences using translator program offered by the JustBio webpage (http://www.justbio.com).

Anti - VCAM -1 mAb had strong affinity and broad cross - species relativity to human and mouse VCAM -1

Following overexpression in E. Coli and purification by anti-HA affinity column chromatography, a 0.3 mg of anti-VCAM-1 Fab was finally obtained from 1 L of a shaking culture. The purity of the antibody was confirmed by SDS-PAGE and Coomassie blue staining (data not shown). Next, using ELISA (Fig. 1A) and western blotting (Fig. 1B), we first tested the reactivity of the antibody to recombinant hVCAM-1 and mVCAM-1. The results indicated that anti-VCAM-1 mAb had cross-species reactivity to hVCAM-1 and mVCAM-1. Furthermore, the kinetic rate constants parameters for interaction between the mAb and VCAM-1 were determined using BIAcore biosensor analysis (Table. 1). The result showed that anti-VCAM-1 mAb has K _D =1.35±0.02x10^-8 of hVCAM-1 and K _D =4.78±0.06x10^-10 of mVCAM-1 respectively.

Table 1

In order to verify the reactivity of the antibody to native VCAM-1, HAECs, HUVECs, and MVECs cultured in the absence or presence of hTNFa or mTNFa were subjected to flow cytometry (Fig. 1C). The result also shows that this mAb could recognize native VCAM-1 expressing on activated human and mouse endothelial cells.

In summary, these findings provide clear evidences that this mAb has strong affinity and broad cross-species reactivity to both hVCAM-1 and mVCAM-1.

Anti - VCAM -1 mAb specifically recognized the sixth IgG - like domain of VCAM -1

To identify an epitope region for the anti-VCAM-1 mAb, we first generated Fc fusions of wild-type and C-terminal serial deletion mutants of human and mouse VCAM-1 extracellular domains (Fig. 2A). Then, following overexpression in HEK293F and affinity purification with protein A sepharose, the equal amounts of purified Fc fusion proteins were subjected to immunoblot analysis for eiptope mapping. Here, anti-human Fc-HRP was used to detect the equal quantity of each Fc fusion proteins. As shown Fig. 2B and 2C, this mAb specifically recognized the sixth IgG-like domain of hVCAM-1 and mVCAM-1 respectively.

The sixth IgG - like domain of VCAM -1 was important for transmigration of U937 cells across activated HUVECs , regardless of interaction between a4b1 integrin and VCAM -1

To investigate the role of the sixth IgG-like domain of VCAM-1 on the transmigration of leukocyte, HUVECs plated on the transwell were incubated in the absence or presence of hTNFa?. Then, transendothelial cell migration assay was performed with U937 human monocytic cell lines in the absence or presence of anti-VCAM-1 Fab. As shown in Fig. 3, we found that anti-VCAM-1 mAb could significantly inhibit the transmigration of U937 cells across activated HUVECs.

To further verify the role of the sixth IgG-like domain on VCAM-1-a4b1 interaction, we first generated vector- or hVCAM-1-overexpressing HEK 293 cell lines (Fig. 4A). Then, we performed neutralizing assay with those HEK293 cell lines, HAEC, and HUVEC and CSFE-labeled U937 in the absence or presence of anti-VCAM-1 mAb. As shown in Fig. 4B-4C, we found that this mAb little affect the VCAM-1-mediated binding of leukocytes to activated endothelial cells.

In summary, these results indicated that the sixth IgG-like domain of VCAM-1 is important for transmigration of U937 cells across activated HUVECs, regardless of interaction between a4b1 integrin and VCAM-1.

The sixth IgG - like domain of VCAM -1 could be a new molecular target for alleviating mouse allogeneic transplantation rejection

In order to investigate the role of the sixth IgG-like domain of VCAM-1 in vivo, we first overproduced anti-VCAM-1 full-IgG from CHO-DG44 stably overexpressing anti-VCAM-1 full-IgG. Here, we confirmed that anti-VCAM-1 full-IgG has the same epitope and cross-species reactivity with anti-VCAM-1 Fab (data not shown). Then, we also established mouse islet allograft models. In detail, 250 IEQ of donor BALB/c male mice (H-2^d) was engrafted under the beneath of left kidney capsule in C57BL/6j (H-2^b) male recipient mice rendered Diabetes Mellitus by Streptozotocin. Then, we treated C57BL/6j (H-2^b) recipient mice with buffer (negative control) or purified anti-VCAM-1 full-IgG daily via intraperitoneal injection up to 7 days (Fig. 5A) and monitored blood glucose level every day until 31 days (Fig. 5B). The result showed that in anti-VCAM-1 full-IgG-treated groups, the blood glucose level has been kept to be perfectly normal, lower than 250 mg/dl, until 31 days post transplantation, but in buffer-treated groups, the blood glucose level gradually became to be abnormal, higher than 250 mg/dl.

In summary, the result shows that the sixth IgG-like domain of VCAM-1 may be a new molecular target for alleviating mouse allogeneic transplantation rejection

According to the present invention, it is expected that it will be possible not only to screen materials that can inhibit the transmigration of leukocyte across endothelial cell without affecting the binding of leukocyte to endothelial cell, but also to effectively prevent or treat VCAM-1 mediated-diseases.

Claims (12)

1. A method for inhibiting the transmigration of leukocyte across endothelial cell by inhibiting activity of the sixth immunoglobulin-like domain of vascular cell adhesion molecule-1 (VCAM-1).
2. The method according to claim 1, wherein the sixth immunoglobulin-like domain of VCAM-1 is derived from human and comprises amino acid sequence of SEQ NO. 1.
3. The method according to claim 2, wherein the sixth immunoglobulin-like domain of VCAM-1 is encoded by nucleotide of SEQ NO. 2.
4. The method according to claim 1, wherein the sixth immunoglobulin-like domain of VCAM-1 is derived from mouse and comprises amino acid sequence of SEQ NO. 3.
5. The method according to claim 4, wherein the sixth immunoglobulin-like domain of VCAM-1 is encoded by nucleotide of SEQ NO. 4.
6. The method according to claim 1, wherein the method does not affect the binding of leukocyte across activated endothelial cell.
7. The method according to claim 1, wherein the inhibiting activity of the sixth immunoglobulin-like domain of vascular cell adhesion molecule-1 (VCAM-1) is accomplished by one of modulators including an oligonucleotide, a chemical compound, and an antibody against the sixth immunoglobulin-like domain of vascular cell adhesion molecule-1 (VCAM-1).
8. A method of preventing or treating VCAM-1 mediated-diseases by inhibiting activity of the sixth immunoglobulin-like domain of VCAM-1.
9. The method according to claim 8, wherein the VCAM-1 mediated-diseases is at least one disease selected from the group consisting of cancers, allergic responses, atherosclerosis, cardiovascular disease, HIV (human immunodeficiency virus, AIDS) disease, arthritis, pneumonia, hypercholesterolemina, sepsis, dermatitis, psoriasis, Crohn's disease, cystic fibrosis, post transplantation late and chronic solid organ rejection, cell or islet transplantation rejection, multiple sclerosis, systemic lupus erythematosis, Graves' disease, thrombotic disease, inflammatory bowel diseases, autoimmune diabetes, diabetic retinopathy, rhinitis, ischemia- reperfusion injury, post-angioplasty restenosis, osteomyelitis, cold, influenza virus disease, chronic obstructive pulmonary disease (COPD), glomerulonephritis, Graves disease, gastrointestinal allergies, sickle cell disease, and conjunctivitis.
10. The method according to claim 8, wherein the method does not inhibit the binding of leukocyte to endothelial cell and the method inhibits only the transmigration of leukocyte across endothelial cell.
11. A kit for screening materials treating or preventing VCAM-1 mediated-diseases, comprising the sixth immunoglobulin-like domain of VCAM-1.
12. A method of screening VCAM-1 mediated-diseases treatment or prevention materials, comprising the steps of:

    (1) contacting a patient of VCAM-1 mediated-diseases patient with a sample;

    (2) detecting increase or decrease of activity of the sixth immunoglobulin-like domain of VCAM-1 expressed on activated endothelial cell; and

    (3) determining the sample that inhibits activity as a VCAM-1 mediated-diseases treatment or prevention materials.

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